Chromatoid body mediated RNA regulation in mouse male germline

Male germ cell differentiation, spermatogenesis is an exceptional developmental process that produces a massive amount of genetically unique spermatozoa. The complexity of this process along with the technical limitations in the germline research has left many aspects of spermatogenesis poorly understood. Post-meiotic haploid round spermatids possess the most complex transcriptomes of the whole body. Correspondingly, efficient and accurate control mechanisms are necessary to deal with the huge diversity of transcribed RNAs in these cells. The high transcriptional activity in round spermatids is accompanied by the presence of an uncommonly large cytoplasmic ribonucleoprotein granule, called the chromatoid body (CB) that is conjectured to participate in the RNA post-transcriptional regulation. However, very little is known about the possible mechanisms of the CB function. The development of a procedure to isolate CBs from mouse testes was this study’s objective. Anti-MVH immunoprecipitation of cross-linked CBs from a fractionated testicular cell lysate was optimized to yield considerable quantities of pure and intact CBs from mice testes. This protocol produced reliable and reproducible data from the subsequent analysis of CB’s protein and RNA components. We found that the majority of the CB’s proteome consists of RNA-binding proteins that associate functionally with different pathways. We also demonstrated notable localization patterns of one of the CB transient components, SAM68 and showed that its ablation does not change the general composition or structure of the CB. CB-associated RNA analysis revealed a strong accumulation of PIWI-interacting RNAs (piRNAs), mRNAs and long non-coding RNAs (lncRNAs) in the CB. When the CB transcriptome and proteome analysis results were combined, the most pronounced molecular functions in the CB were related to piRNA pathway, RNA post-transcriptional processing and CB structural scaffolding. In addition, we demonstrated that the CB is a target for the main RNA flux from the nucleus throughout all steps of round spermatid development. Moreover, we provided preliminary evidence that those isolated CBs slice target RNAs in vitro in an ATPdependent manner. Altogether, these results make a strong suggestion that the CB functions involve RNA-related and RNA-mediated mechanisms. All the existing data supports the hypothesis that the CB coordinates the highly complex haploid transcriptome during the preparation of the male gametes for fertilization. Thereby, this study provides a fundamental basis for the future functional analyses of ribonucleoprotein granules and offers also important insights into the mechanisms governing male fertility.Miesten sukusolujen kehittyminen, spermatogeneesi, on tärkeä ja monella tavoin poikkeuksellinen kehitysprosessi, joka tuottaa suunnattomia määriä geneettisesti yksilöllisiä siittiöitä. Spermatogeneesi on monimutkainen, tarkasti säädelty tapahtumasarja, mikä myös aiheuttaa teknisiä haasteita spermatogeneesin molekyylimekanismien tutkimisessa. Näin ollen prosessin yksityiskohdat ovat vielä pitkälti tuntemattomia. Eräs siittiön haploidien esiasteiden (pyöreät spermatidit) erityisominaisuus on niiden ainutlaatuisen runsas transkriptionaalinen aktiivisuus. Genomin aktiivinen ilmentyminen puolestaan edellyttää tehokkaita ja täsmällisiä RNA:n säätelymekanismeja. Pyöreiden spermatidien solulimassa sijaitsee epätavallisen suuri RNA:ta ja proteiineja sisältävä rakenne, kromatoidikappale (chromatoid body, CB), joka ilmaantuu juuri voimakkaimman transkriptioaallon aikana ja osallistuu RNA-säätelyyn. Tutkimuksen tavoitteena oli selvittää CB:n toimintaa siittiönkehityksen aikana. Tärkeänä osana tutkimusta kehitimme menetelmän, jonka avulla CB:t voidaan eristää hiiren kiveksestä. Menetelmä on yksinkertainen, nopea ja tehokas, ja sen avulla saadaan eristettyä toistettavasti rakenteeltaan ehjiä CB:ta, joiden puhtaus on hyvä ja määrä riittävä molekyylitason analyysiin. Jatkotutkimukset paljastivat, että suurin osa CB:n sisältämistä proteiineista on erilaisilla RNA-säätelyreiteillä toimivia RNA:ta sitovia proteiineja. Useat CB:n proteiineista pysyvät rakenteessa stabiilisti, mutta näytimme myös, että RNA:ta sitova proteiini SAM68 vierailee CB:ssa vain hyvin hetkellisesti tarkasti määritellyssä kehitysvaiheessa. Poistogeenisen hiirimallin avulla saimme selville, ettei SAM68 proteiinia kuitenkaan tarvita CB:n muodostumiseen. Tulostemme mukaan CB:een kulkeutuu suuri määrä RNA:ta kaikissa pyöreiden spermatidien kehitysvaiheissa. RNA-sekvenointi osoitti, että pienet piRNA (PIWI-interacting RNA) molekyylit ovat rikastuneet CB:ssa. Lisäksi CB sisältää suuren joukon erilaisia lähetti-RNA:ita ja aivan tuntemattomia intergeenisiä eikoodaavia RNA:ita. Analyysimme mukaan hallitsevin CB:n molekyylireiteistä on piRNAvälitteinen RNA-säätelyreitti, mutta myös lähetti-RNA:n prosessointiin liittyvät tekijät ovat vahvasti edustettuina. CB ei selvästikään ole vain passiivinen RNA:n varastointipaikka, vaan näytimme sen kykenevän ATP:stä riippuvaiseen RNA prosessointiin in vitro Kaikki tutkimuksen tulokset osoittavat, että CB on keskeinen RNA:n säätelykeskus, joka koordinoi sukusolujen erittäin monimuotoista transkriptomia. Tällä toiminnallaan CB osallistuu tärkeänä tekijänä miesten hedelmällisyyden ja sukusolujen geneettisen ja epigeneettisen informaation säätelyyn.Siirretty Doriast

piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis.

MIWI catalytic activity is required for spermatogenesis, indicating that piRNA-guided cleavage is critical for germ cell development. To identify meiotic piRNA targets, we augmented the mouse piRNA repertoire by introducing a human meiotic piRNA cluster. This triggered a spermatogenesis defect by inappropriately targeting the piRNA machinery to mouse mRNAs essential for germ cell development. Analysis of such de novo targets revealed a signature for pachytene piRNA target recognition. This enabled identification of both transposable elements and meiotically expressed protein-coding genes as targets of native piRNAs. Cleavage of genic targets began at the pachytene stage and resulted in progressive repression through meiosis, driven at least in part via the ping-pong cycle. Our data support the idea that meiotic piRNA populations must be strongly selected to enable successful spermatogenesis, both driving the response away from essential genes and directing the pathway toward mRNA targets that are regulated by small RNAs in meiotic cells.This work was supported by the National Institutes of Health R37 grant GM062534-14 to G.J.H. iTRAQ was performed with assistance from the Cold Spring Harbor Laboratory Proteomics Shared Resource, which is supported by Cancer Center support grant 5P30CA045508. W.S.S.G. is a McClintock Fellow of the Watson School of Biological Sciences and is supported by the NSS Scholarship from the Agency for Science, Technology and Research, Singapore. O.H.T. is supported by a fellowship of the Human Frontier Science Program. R.B. is supported by the Starr Centennial Scholarship from the Watson School of Biological Sciences. G.J.H. is a Howard Hughes Medical Institute Investigator.This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via http://dx.doi.org/10.1101/gad.260455.11

The RNA Binding Protein SAM68 Transiently Localizes in the Chromatoid Body of Male Germ Cells and Influences Expression of Select MicroRNAs

The chromatoid body (CB) is a unique structure of male germ cells composed of thin filaments that condense into a perinuclear organelle after meiosis. Due to the presence of proteins involved in different steps of RNA metabolism and of different classes of RNAs, including microRNAs (miRNAs), the CB has been recently suggested to function as an RNA processing centre. Herein, we show that the RNA binding protein SAM68 transiently localizes in the CB, in concomitance with the meiotic divisions of mouse spermatocytes. Precise staging of the seminiferous tubules and co-localization studies with MVH and MILI, two well recognized CB markers, documented that SAM68 transiently associates with the CB in secondary spermatocytes and early round spermatids. Furthermore, although SAM68 co-immunoprecipitated with MVH in secondary spermatocytes, its ablation did not affect the proper localization of MVH in the CB. On the other hand, ablation of the CB constitutive component MIWI did not impair association of SAM68 with the CB. Isolation of CBs from Sam68 wild type and knockout mouse testes and comparison of their protein content by mass spectrometry indicated that Sam68 ablation did not cause overall alterations in the CB proteome. Lastly, we found that SAM68 interacts with DROSHA and DICER in secondary spermatocytes and early round spermatids and that a subset of miRNAs were altered in Sam68−/−germ cells. These results suggest a novel role for SAM68 in the miRNA pathway during spermatogenesis

Dicer1 Depletion in Male Germ Cells Leads to Infertility Due to Cumulative Meiotic and Spermiogenic Defects

Background: Spermatogenesis is a complex biological process that requires a highly specialized control of gene expression. In the past decade, small non-coding RNAs have emerged as critical regulators of gene expression both at the transcriptional and post-transcriptional level. DICER1, an RNAse III endonuclease, is essential for the biogenesis of several classes of small RNAs, including microRNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), but is also critical for the degradation of toxic transposable elements. In this study, we investigated to which extent DICER1 is required for germ cell development and the progress of spermatogenesis in mice.Principal Findings: We show that the selective ablation of Dicer1 at the early onset of male germ cell development leads to infertility, due to multiple cumulative defects at the meiotic and post-meiotic stages culminating with the absence of functional spermatozoa. Alterations were observed in the first spermatogenic wave and include delayed progression of spermatocytes to prophase I and increased apoptosis, resulting in a reduced number of round spermatids. The transition from round to mature spermatozoa was also severely affected, since the few spermatozoa formed in mutant animals were immobile and misshapen, exhibiting morphological defects of the head and flagellum. We also found evidence that the expression of transposable elements of the SINE family is up-regulated in Dicer1-depleted spermatocytes.Conclusions/Significance: Our findings indicate that DICER1 is dispensable for spermatogonial stem cell renewal and mitotic proliferation, but is required for germ cell differentiation through the meiotic and haploid phases of spermatogenesis

SAM68 co-immunoprecipitates with MVH in secondary spermatocytes.

<p>(<b>A</b>) Total extract from secondary spermatocytes were immunoprecipitated with an anti-SAM68 antibody and analysed in Westeern blot with anti-MVH and anti-Sam68 antibodies. (<b>B</b>) Cellular extracts from spermatocytes (I sp.cytes), secondary spermatocytes (II sp.cytes) and round spermatids (sp.tids) were immunoprecipitated with an anti-SAM68 antibody and detected with anti-MVH antibody. Western blot analysis shows a specific interaction of the two proteins in secondary spermatocytes, while no signal is detected in other germ cell populations.</p

Isolation and analysis of chromatoid bodies from male germ cells.

<p>(<b>A</b>) Schematic representation of the CB isolation protocol. (<b>B</b>) Immunofluorescence analysis of different steps of CB immunoprecipitation experiment, comparing <i>Sam68</i> wild type (WT) and knockout (KO) extracts. Cells were stained with an anti-MVH antibody (red) and with Hoechst to detect nuclei. CELLS  =  cells before lysis; LYS  =  lysate; PEL  =  pellet fraction; P2F  =  pellet fraction after filtration. (<b>C</b>) Immunoblotting of the CB extracts with anti-MVH antibody to validate the success of the purification. The less intensive signal in the knockout CB fraction indicates the lower number of CBs isolated from the knockout testes compared to the control. anti-ACR, negative control IP; anti-MVH, CB IP. (<b>D</b>) RNA gel to demonstrate the presence of piRNAs. Total RNA was extracted from the the lysate (LYS) and CB IPs (CB), radiolabelled and run into a polyacrylamide gel in. (<b>E</b>) Mascot analysis of the main CB components. All major CB proteins were present in the knockout CBs. The uniprot entry name KDHR_MOUSE equals to SAM68.</p

Co-localization of SAM68 with MVH and MILI in male germ cells.

<p>(<b>A</b>) Isolated male germ cells were co-stained with an anti-SAM68 antibody (red), an anti-MVH antibody (green) and with Hoechst (blue) to detect nuclei. SAM68 and MVH partially co-localize in the CB of secondary spermatocytes (arrows), while in primary spermatocytes SAM68 is nuclear and MVH is cytoplasmic, and in round spermatids SAM68 is nuclear and MVH is predominantly localized in the CB. (<b>B</b>) Isolated germ cells were analysed by immunofluorescence using the anti-SAM68 antibody (red) and the anti-MILI antibody (green). Nuclei were stained with Hoechst (blue) to identify cell stages by nuclear morphology. In primary spermatocytes SAM68 localizes in the nucleus, while MILI is cytoplasmic; in round spermatids SAM68 is nuclear and MILI is absent. The localization of the two proteins partially overlaps only in the CB of secondary spermatocytes.</p